Theor Appl Genet (1984) 68:515-519

9 Springer-Verlag 1984

Genetic instability in protoclones of potato (Solanum tuberosum L. cv. 'Bintje'): new types of variation after vegetative propagation K. Sree Ramulu, P. Dijkhuis and S. Roest Research Institute 1TAL, P.O. Box 48, NL-6700 AA Wageningen, The Netherlands Received January 31, 1984; Accepted June 16, 1984 Communicated by G. Wenzel

Summary. The transmission of variation from protoplast-derived plants o f tetraploid potato cultivar 'Bintje' to tuber progeny was examined. The morphological alterations of a majority of the variant protoclones were transmitted to corresponding tuber progeny. Some o f the normal and variant protoclones gave new phenotypes, or segregated into parental and new phenotypes after vegetative propagation. The ploidy levels o f almost all these clones remained unchanged after propagation. It was concluded that the occurrence o f variation after vegetative propagation was due to somatic segregation of chimeras resulting from gene mutations or chromosome structural rearrangements in only part o f the regenerated plant. The origin of variation is discussed in the light of these results.

Key words: Potato protoclones - Transmission of variation - Chimeras - Somatic mutations

Introduction Non-meristematic tissue explants, cells or protoplasts removed from the stabilizing environment of the intact plant and cultured in vitro, display genetic instabilities o f various kinds. These include gross chromosome variation (polyploidy, aneuploidy), structural changes in chromosomes and gene mutations. It is evident from several studies that these changes are transmitted to regenerated plants and to their progeny ( D ' A m a t o 1975, 1978; Bayliss 1980; Larkin and Scowcroft 1981; Sree R a m u l u 1982; Browers and Orton 1982; Barbier and Dulieu 1983; L r r z and Scowcrort 1983). The variation occuring during the plant regeneration process stimulated interest in recent years, both from the point of

using it in plant breeding, and because it may be undesirable when stable reproduction of a specific genotype is essential, as in the case of micropropagation and in genetic transformation studies. In potato, a wide range of variation in morphological characters, disease resistance and ploidy levels has been reported among plants regenerated from protoplasts or tissue cultures of several genotypes, including the Dutch cv. 'Bintje' (Wenzel et al. 1979; Behnke 1979, 1980; Shepard et al. 1980; Wenzel and Uhrig 1981; Van Harten etal, 1981; Jacobsen 1981; Thomas et al. 1982; Karp et al. 1982; Austin and Cassells 1983; Sree Ramulu et al. 1983). 'Bintje' is the most important cultivar in the Netherlands because of its excellent quality for domestic and industrial uses. However, it is susceptible to most potato diseases. The conventional breeding methods have not been successful until now due to male sterility and heterozygosity of the cultivar. Molecular and in vitro somatic approaches might be useful in this regard. However, this requires the development of cultures which could give efficient and stable regeneration of plants. Recently, using the method of protoplast isolation and culture described for dihaploid clones of potato (Binding et al. 1978), plants have been regenerated from shoot culture-derived protoplasts of cv. 'Bintje' and analysed for variation (Bokelmann and Roest 1983; Sree Ramulu et al. 1983). This work was undertaken in order to evaluate stability among regenerated plants. The present article reports new results on the occurrence of different types o f variation after vegetative propagation of potato (cv. 'Bintje') protoclones.

Materials and methods Plant material

The cultivar 'Bintje' of tetraploid Solanum tuberosum (2n=4x=48) was used. The method of protoplast isolation and culture described for dihaploid potato clones (Binding et al. 1978) was used with some modifications. For details on the procedures of propagation of shoot cultures, protoplast culture and plant regeneration, reference is made to Bokelmann and Roest (1983).

516

K. Sree Ramulu et al.: Genetic instability in protoclones of potato

Morphological characterization The morphological characterization described previously (Sree Ramulu et al. 1983) for protoplast-derived plants (protoclones) of cv. 'Bintje' was followed in this study to analyse the tuber progeny. The term "protoclone" is used to denote each individual shoot or plant obtained from protoplast regeneration process. Thus, a given protoplast-callus can produce one or more protoclones. The term is not intended to suggest identity with the mother plant cv. 'Bintje', as variation was found among protoclones (obtained from the same or different protoplast-callus) as well as within a protoclone. The growth and vigour of plants, leaf characters such as colour, shape, texture and size and axillary branching were studied. The characters of each of the plants were scored several times during the various periods of growth and compared with those of the control plants. They were classified into three types according to their morphological characters, 1) normal-looking plants (resembling the mother plant cv. 'Bintje'), 2) plants showing variation in a few (1-3) characters and 3) plants showing variation in several (> 3) characters (gross aberrants). All the plants were grown under greenhouse conditions. The number of tubers grown from each regernated plant (protoclone) varied from 1-20, it being 3 in most cases. Thus, more than 2,000 tuber-propagated plants were scored from 613 protoclones.

Cytological analysis To determine ploidy levels of plants, root-tips collected from young potted plants were pretreated with 8-hydroxy-quiniline (0.002 M) for 4 h at 18 ~ and then fixed in absolute alcohol: glacial acetic acid (3 : 1 v/v) for 24 h. The roots were hydrolysed in 1 N HC1 for 8 min at 60~ and stained in Feulgen. Chromosome counts greater than 50 were _+2 chromosomes. Results The data on the types o f protoclones and their tuber progeny are given in Table 1. There were altogether 391 n o r m a l - l o o k i n g protoclones. After tuber propagation, 373 resembled the normal type, while the remainder (18) showed variation: 14 protoclones showed new phenotypes and 4 segregated each into new and n o r m a l phenotypes. In all, 11 different types occurred, each varying in one or a few morphological characters.

Out o f 125 protoclones showing variation in a few characters, 96 resembled the parental types (i.e. parentprotoclones) in the next vegetative generation, 13 reverted to normal type and 16 segregated into parental types and new or n o r m a l types (Table 1). The new phenotypes (12 different types) varied in one or a few traits. A feature of interest was the tuber progeny o f a chlorophyll chimera with a small yellow sector on one o f the leaves. The progeny segregated into 1 n o r m a l green and 2 variegated (chimeric) plants, all being tetraploid and normal in other morphological characters. One of the chimeric plants (Fig. 1A) consisted of 5 shoots: 2 chimeric, 1 completely yellow shoot and 2 n o r m a l green. The other chimeric plant (Fig. 1 B) had four shoots, all being chimeric. Tubers raised from the former plant gave 1 completely yellow plantlet (Fig. 1 C), 1 chimera with completely yellow and green shoots (Fig. 1 D) and 2 completely green plants (not shown in Fig. 1). As the yellow plant is weak and slow growing, it is being propagated in vitro in order to use as a m a r k e r in protoplast fusion and transformation studies. The other 103 protoclones were grossly aberrant in phenotype, 61 showing chimeric structures within the plant, i.e. chlorotic spots or sectors o f different size and colour on leaves and morphologically a b n o r m a l and normal shoots. A high frequency (ca. 80%) o f the protoclones had altered ploidies (aneuploid, octoploid, mixoploid). After vegetative propagation, 99 protoclones resembled the respective parental type, while 4 showed new phenotypes differing in one or more traits (Table 1). Thus, in the total material 18 normal-looking protoclones and 20 variant protoclones segregated or showed new phenotypes after vegetative propagation. They were derived from 35 different protoplast-calli. Each o f these calli also gave rise to one or more other protoclones (normal or variant) which did not segregate after tuber propagation.

Table 1. Types ofprotoclones (regenerated plants) and their tuber progeny in tetraploid potato cv. 'Bintje' (2n = 4x = 48) Protoclones Type

Tuber progeny No.

%

Types Normal

N ormal-looking Varying in a few characters Varying in several characters (gross aberrants) Total

Parental New 14

New+ normal

New+ Normal+ parental parental 2 -

3

2

3

391 125 103

63.2 20.2 16.6

373 13 -

96 99

4

4 11 -

619

100.0

386

195

18

15

Normal and parental types refer to plants resembling cv. 'Bintje' and variant protoclones, respectively

K, Sree Ramulu et al.: Genetic instability in protoclones of potato

517

Fig. 1 A-D. Tuber progeny from a protoplast-derived chlorophyll chimera in tetraploid potato cv. 'Bintje'. A variegated plant with five shoots: 1 and 2 chimeric shoots, 3 a completely yellow shoot, 4 and 5 green shoots; B variegated plant with four chimeric shoots; C a completely yellow plantlet; D a chimeric plantlet with completely yellow and green shoots, both derived from the tubers of plant A

Table 2 gives data on chromosome numbers of all the 38 segregating protoclones and their tuber progeny. All 18 normal-looking protoclones and their progeny (new and normal types) were tetraploid (2n = 48). Among the protoclones showing variation in a few or several characters, there were not only tetraploids, but also aneuploids, mixoploids and an octoploid. After tuber propagation, the chromosome numbers of all the protoclones remained the same, except in 2 mixoploids which segregated into different ploidy-types. One of the mixoploid clones ("89-J") gave 12 progeny plants of 6 different phenotypes, each varying in one or a few mor-

phological characters (Table 3). Four of these were mixoploids, one was tetraploid and one octoploid. Discussion The results obtained show that the morphological alterations of a majority of the variant protoclones were transmitted to the tuber progeny. Furthermore, the analysis of the tuber progeny revealed the following features. 1) Some of the normal and variant protoclones segregated or showed new phenotypes after vegetative

518

K. Sree Ramulu et al.: Genetic instability in protoclones of potato

Table 2. Chromosome numbers (in parentheses) of segregating protoclones and their tuber progenies in tetraploid potato cv. 'Bintje' (2n = 4x = 48) Distribution of tuber progeny plants a showing different phenotypes

Protoclones Type

No.

Normal

Normal-looking

4 (48) 14 (48) 2 (48) 5 (48) 5 (46) 1 (48, 96) b 2 (48)

8 (48)

Varying in a few characters

1 (45)

Varying in several characters (gross aberrants)

Parental

New

3 (48)

4 (48) 35 (48) 2 (48) 7 (48) 6 (46) 2 (48, 96) b

5 (48) 9 (46) 1 (48) 3 (48)

2 (48)

2 (45)

1 (45)

1 (96) 2 (47)

8 (96) 6 (47) 12~

1 (48, 94, 96) ~

No. of tuber progeny plants per protoclone ranged from 2-12, it being mostly 3 b Mixoploid ~ Mixoploid clone "89-J" giving 12 tuber progeny plants (for details on chromosome numbers, see Table 3)

Table 3. Phenotypes and chromosome numbers of tuber progeny obtained from protoclone "89-J" in tetraploid potato cv. 'Bintje' (2n = 4x = 48). Aneuploid chromosome numbers are reported in parentheses Pheno- No. of plants ~ Distribution of mitotic cells with types analysed in chromosome numbers as grouped each phenotype below 48 (4x) 1

3

5

2

2

4

3 4 5 6

1 2 3 1

8 7 -

48-95

96 (8x)

1 (50), 3 (52),

6

2 (83), 2 (92) 2 (60), 3 (72), 2 (85) 5 (91), 4 (94) -

5 4 8 7

a All plants ofphenotypes 1, 2, 4 and 5 were mixoploid

p r o p a g a t i o n (within-clone variation). The new phenotypes differed in one or a few traits. 2) Most o f the segregating protoclones were derived from different calli which also p r o d u c e d one or more non-segregating protoclones (normal or variant). 3) The gross-aberrant protoclones were associated, in general with p l o i d y alteration. However, this does not a p p e a r to be the source o f variation found after vegetative propagation, because the ploidies o f almost all the protoclones r e m a i n e d u n c h a n g e d after p r o p a g a t i o n and the progenies differing in p h e n o t y p e had the same p l o i d y level.

These results suggest that variation a m o n g the vegetatively p r o p a g a t e d plants m a y be due to somatic segregation of chimeras resulting from gene mutations or chromosome structural rearrangements in only part of the regenerated plant. In tetraploid potato, chromosomal a n d gene alterations can be tolerated because o f the buffering capacity o f the polyploid condition, as in the case o f tobacco and wheat (Ogura 1976; A s h m o r e and G o u l d 1981; Armstrong et al. 1983). Some of the types of variation found among the tuber progeny include dark green, virescent, tiny dwarf, purple sprouts, wildings and foliage types such as narrow, simple, pinnate, spinach, glabrous and blistered. Genetical investigations in dihaploid potato suggest that such alterations are caused by mutations (Howard 1970). Similarly, variegated plants have been studied in dihaploid potato (Klopfer 1965). These studies indicate that mutant plastomes were carried in the histogenetic L2 layer and that they exhibited maternal inheritance. Due to difficulties in cv. 'Bintje', of performing sexual crosses (male sterility), and because of heterozygous nature of the tetraploid potato genome, it is not possible to perform genetic analysis at present. Advances in molecular methods (DNA probes, isozyme characteristics, banding techniques) might help in obtaining additional information in this regard. As to the source of chimerism in the regenerants (protoclones) which segregated after tuber propagation, the most likely explanation is that they are initially derived from more than one cell. A high frequency of protoplast-derived calli comprised heterogeneous populations of cells as a consequence of instability of the mitotic process (abnormal DNA synthesis and mitosis) (Sree Ramulu et al. 1984). Therefore, it is likely that in the case of chimeras, one or more genetically different (mutated) cells participate in the organization of an adventitious bud. Previously, evidence for the multicellular origin of adventitious buds and plantlets in vitro has been obtained in the case of morphological and cytological chimeras, including mixoploids (chromosome mosaics) (Ogura 1976; Bennici 1976; Sree Ramulu et al. 1976; Mix et al. 1978; Bennici and D'Amato 1978; Browers and Orton 1982).

K. Sree Ramulu et al.: Genetic instability in protoclones of potato Several factors, such as genetic architecture of species or genotype, explant origin, composition of the medium and culture age are associated with genetic instability during the plant regeneration process (Torrey 1967; D'Amato 1975; Wenzel et al. 1979; Larkin and Scowcroft 1981; Evans and Gamborg 1982). Therefore, appropriate conditions have to be established in order to reduce or avoid undesirable gross aberrants and chimeras. This is of practical importance for the proper utilization of genetic manipulation techniques and to exploit the most promising source of somaclonal variation for crop improvement, i.e. point mutations. The a p p e a r a n c e o f p r o g e n y (tuber) plants varying in a few characters which were derived from completely n o r m a l - l o o k i n g protoclones, stresses the need o f a greater insight into the chimeric fine structure o f protoplast-calli. A note on the geneology in regards to the segregation in vegetatively p r o p a g a t e d plants and chimeric composition o f the protoplast-calli is in progress. Acknowledgements. Several suggestions for improvement of the manuscript were given by Prof. B. de Groot and Prof. J. Sybenga. References Armstrong KC, Nakamura C, Keller WA (1983) Karyotype instability in tissue culture regenerants of triticale (x Triticoseeale Wittmack) cv. 'Welsh' from 6-month-old callus cultures. Z Pflanzenzticht 91 : 233-245 Ashmore SE, Gould AR (1981) Karyotypic evolution in a turnout derived plant tissue culture analysed by Giemsa C-banding. Protoplasma 106:297-308 Austin S, Cassells AC (1983) Variation between plants regenerated from individual calli produced from separated potato stem callus cells. Plant Sci Lett 31: 107-114 Barbier M, Dulieu HL (1983) Early occurrence of genetic variants in protoplast cultures. Plant Sci Lett 29:201-206 Bayliss MW (1980) Chromosomal variation in plant tissues in culture. Int Rev Cytol (Suppl) 11 A: 113-144 Behnke M (1979) Selection of potato callus for resistance to culture filtrates of Phytophthora infestans and regeneration of resistant plants. Theor Appl Genet 55:69-71 Behnke M (1980) Selection of dihaploid potato callus for resistance to the culture filtrates of Fusarium oxysporum. Z Pflanzenziieht 85:254-258 Bennici A (1976) Cytological analysis of roots, shoots, and plants regenerated from suspensions and solid in vitro cultures of haploid Pelargonium. Z Pflanzenziicht 72:199-205 Bennici A, D'Amato F (1978) In vitro regeneration of durum wheat plants. 1. Chromosome numbers of regenerated plantlets. Z Pflanzenziicht 83:305-311 Binding H, Nehls R, Schieder O, Sopory SK, Wenzel G (1978) Regeneration of mesophyll protoplasts isolated from dihaploid clones of Solanum tuberosum. Physiol Plant 43: 52-54 Bokelmann GS, Roest S (1983) Plant regeneration from protoplasts of potato (Solanum tuberosum cv. 'Bintje'). Z Pflanzenphysiol 109:259-265 Browers MA, Orton TJ (1982) Transmission of gross chromosomal variability from suspension cultures into regenerated celery plants. J Hered 73:159-162 D'Amato F (1975) The problem of genetic stability in plant tissue and cell cultures. In: Frankel O, Hawkes JG (eds) Crop genetic resources for today and tomorrow. Cambridge Univ Press, London, pp 333-348

519 D'Amato F (1978) Chromosome number variation in cultured cells and regenerated plants. In: Thorpe TA (ed) Frontiers of plant tissue culture 1978. Calgary, Int Assa Plant Tissue Culture, pp 287-295 Evans DA, Gamborg OL (1982) Chromosome stability of cell suspension cultures of Nicotiana spp. Plant Cell Rep l: 104-107 Harten AM van, Boulter H, Broertjes C (1981) In vitro adventitious bud techniques for vegetative propagation and mutation breeding of potato (Solanum tuberosum L.) 2. Significance for mutation breeding. Euphytica 30:1-8 Howard HW (1970) Genetics of the potato Solanum tuberosum. Logos Press, London Jacobsen E (1981) Polyploidization in leaf callus tissue and in regenerated plants of dihaploid potato. Plant Cell Tissue Organ Culture 1:77-84 Karp A, Nelson RS, Thomas E, Bright SWJ (1982) Chromosome variation in protoplast-derived potato plants. Theor Appl Genet 63: 265-272 Klopfer K (1965) Erfolgreiche experimentelle Entmischungen und Umlagerungen periklinalchim~rischer Kartoffelklone. Ztichter 35:201-214 Larkin PJ, Scowcroft WR (1981) Somaclonal variation - a novel source of variability from cell cultures. Theor Appl Genet 60:197-214 Lrrz H, Scowcroft WR (1983) Variability among plants and their progeny regenerated from protoplasts of Su/su heterozygotes of Nicotiana tabacum. Theor Appl Genet 66:67-76 Mix G, Wilson HM, Forougi-Wehr B (1978) The cytological status of Hordeum vulgare L. regenerated from microspore callus. Z Pflanzenziicht 80:89-99 Ogura H (1976) The cytological chimeras in original regenerates from tobacco tissue cultures and their offsprings. Jpn J Genet 51:161-174 Shepard JF, Bidney D, Shahin E (1980) Potato protoplasts in crop improvement. Science 28:17-24 Sree Ramulu K, Devreux M, Ancora G, Laneri U (1976) Chimerism in Lycopersicon peruvianum plants regenerated from in vitro cultures of anthers and stem internodes. Z Pflanzenzticht 76:299-319 Sree Ramulu K (1982) Genetic instability at the S-locus of Lycopersieon peruvianum plants regenerated from in vitro culture of anthers: generation of new S-specificities and S-allele reversions. Heredity 49:321-332 Sree Ramulu K, Dijkhuis P, Roest S (1983) PhenOtypic variation and ploidy level of plants regenerated from protoplasts of tetraploid potato (Solanum tuberosum L. cv. 'Bintje'). Theor Appl Genet 65:329-339 Sree Ramulu K, Dijkhuis P, Roest S, Bokelmann GS, De Groot B (1984) Early occurrence of genetic instability in protoplast cultures of potato. Plant Sci Lett (in press) Thomas E, Bright SWJ, Franklin J, Gibson RW, Lancaster V, Miflin BJ (1982) Variation amongst protoplast-derived potato plants (Solanum tuberosum cv. 'Marls Bard'). Theor Appl Genet 62:65-68 Torrey JG (1967) Morphogenesis in relation to chromosomal constitution in long term plant tissue cultures. Physiol Plant 20:265 -275 Wenzel G, Schieder O, Przewozny T, Sopory SK, Melchers G (1979) Comparison of single cell culture derived Solanum tuberosum L. plants and a model for their application in breeding programs. Theor Appl Genet 55:49-55 Wenzel G, Uhrig H (1981) Breeding for virus and nematode resistance in potato via anther culture. Theor Appl Genet 59:333-340

Genetic instability in protoclones of potato (Solanum tuberosum L. cv. 'Bintje'): new types of variation after vegetative propagation.

The transmission of variation from protoplast-derived plants of tetraploid potato cultivar 'Bintje' to tuber progeny was examined. The morphological a...
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